EXCEED THE SPACE PROVIDED, This proposal focuses on unresolved issues of early B lineage differentiation in mice and, especially, in humans. Aim 1. During the initial funding period a novel model of human B cell development was characterized, the EU12 cell line, in which CD34*/^ heavy chain" pro-B cells spontaneously undergo step- wise differentiation to become IgMVlgD* B cells. This cell line was used to test the hypothesis that intraclonal V(D)J recombinatoriai diversification contributes to the efficient generation of a primary B cell repertoire and in a preliminary way to define the changes in gene expression profile during B lineage differentiation, (ii) Having been validated as a model for human B lymphopoiesis, carefully dissected EU12 subpopulations will be used in a comprehensive microarray analysis to obtain a more complete transcriptome picture of human B cell development. Selected developmental^ regulated genes will be validated by realtime F'CR and at the protein level, (ii) The gene expression profile of normal sorted human B cell developmental stages will then be elucidated. (Hi) Armed with this comparative data, potentially informative genes will be overexpressed or inhibited to determine the effects on growth and differentiation of EU12 B lineage cells. Aim 2. Toward identifying factors essential for human B lymphopoiesis, the function of novel candidate growlh factors identified in gene expression analysis of human stromal cell lines that do or do not support Blymphopoiesis will be assessed. Recently a transplantation model of human CD34+ stem cells in immunodeficient mice has been shown to allow human B and T cells to develop to the extent of mounting an isotype switchedantigen- specific immune response. A breeding colony of these mice has been initiated to determine (i) if the microenvironment for human B cell development is provided solely by mouse cells or by human stromal cells derived from the transplanted CD34+ cells, and (ii) to define the requirement for mouse cytokines using cytokine gene knockout models. The ultimate goal is to define soluble and cellular factors required for human B cell development. Aim 3. A novel family of Fc receptor related genes, the FcR homo'logs, was discovered during the initial funding period. Each of the first five FcRH members appears to have a unique expression pattern and function during B cell differentiation. Studies in this aim focus on FcRHt, 2:, 4 and 5 in terms of (i) precise expression patterns, (ii) function, and (ill) ligands. The proposed studies should define novel features of B cell differentiation that are likely to have significant clinical implications.